Immortalized Porcine Alveolar Macrophage

ABSTRACT

The present invention relates to immortalized porcine alveolar macrophages (PAMs), to cell cultures comprising such PAMs, to methods for the immortalization of PAMs, to methods of replicating PRRS virus on immortalised PAMs and to methods for the preparation of vaccines comprising PRRSV.

The present invention relates to immortalized porcine alveolarmacrophages (PAMs), to cell cultures comprising such PAMs, to methodsfor the immortalization of PAMs, to methods of replicating PRRS virus onimmortalised PAMs and to methods for the preparation of vaccinescomprising PRRSV.

Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) is by farthe most economically important arterivirus, affecting swine farmingindustries around the world. Infection with this virus results in slowgrowth, decreased feed efficiency, anorexia, and fever in weaning tofinishing pigs, abortion in pregnant sows and respiratory problems inyoung pigs. In the US alone, yearly losses associated with PRRSVinfection were estimated to lie around $560 million in 2005 and $664million in 2011. PRRSV infection ranks as the number one healthchallenge for the pig industry. Considering the emergence of highlyvirulent strains of PRRSV in South-East Asia in 2006 and the fact thatthe Asian swine industry is the largest in the world, it can safely beassumed that losses in this part of the world are even considerablyhigher than those reported for Europe and the US.

PRRSV remains a major threat to the swine industry since the associateddisease has proven to be difficult to control, in spite of theavailability of both live attenuated and killed vaccines against PRRSV.

For PRRSV vaccine production, live attenuated or inactivated, the virusmust be replicated on susceptible cells. One of the problems faced inthe propagation of PRRSV is the highly restricted cell tropism of thevirus. It mainly infects primary porcine alveolar macrophages (PAMs).Such PAMs are difficult to obtain: they are usually obtained throughlung lavage of piglets at usually 6-12 weeks of age (Wensvoort, G. etal., The Veterinary Quarterly 13: 121-130 (1991)). This method iscumbersome, expensive and leads to batches with a high batch-to-batchinconsistency. Moreover, primary PAMs can only be kept in cell culturefor a very limited amount of time. Thus, although primary PAMs are verysuitable for growing PRRSV, they are only useful in e.g. experimentalinfection studies and for making experimental vaccines. Production of acommercial vaccine on primary PAMs is economically not feasible.

Due to this problem, scientists have tried to find other cells orbetter; cell lines, that are susceptible to PRRSV. Only three cell lineshave been identified: MA104 monkey kidney cell line and two derivativesof MA104: MARC-145 and CL2621 cells (Kumar Shanmukhappa et al., VirologyJournal 2007, 4: 62). These cell lines are currently commercially usedfor the propagation of PRRSV.

Although it is possible to grow PRRSV to relatively high titers on thesecell lines, such non-PAM not even being porcine cell lines have adisadvantage: since they are not the natural host of PRRSV, PRRSV has tobe adapted to these cell lines before high titers can be obtained. As aconsequence, neither MA104 nor MARC-145 or CL2621 would be the firstchoice for the propagation of newly discovered field strains. Forpicking up new PRRSV field isolates the natural host cell, the PAM,would be much more suitable.

It has been contemplated that an immortalized PAM cell line couldprovide a solution to the problems identified above.

Immortalized PAM cell lines can in principle be grown without limitationto their passage level and they are the most suitable host cells forPRRSV.

Several attempts have been made to develop immortalized PAM cell lines.

PCT Patent Application WO2008/089094 discloses two natural, deliberatelynon-transformed PAM mutants obtained from porcine fetuses. These cellsshow immortalized PAM cell line behavior and they are capable of growingPRRSV. The disadvantage of the method described is, that it is atrial-and-error based method of which the outcome is highly uncertain.Specifically it is questionable if any fetal cell line isolated usingthis method is indeed immortalized in a stable manner or merely has asomewhat extended life span or is capable of dividing just a few moretimes when compared to a primary PAM. The method is thus unattractive tothe skilled person in need of an immortalized PAM cell line.

A Thesis by Jian-Jun Jia (August 2009, Universite de Montreal, Montreal,Canada) describes an allegedly porcine lung cell line susceptible toPRRSV, but this cell line is not an alveolar but an epithelial cell lineand furthermore later turned out not to be of porcine origin (D. W.Silversides et al., Journal of virology 84: 5454-5455 (2010)).

A further attempt to make a genuine immortalized PAM cell line isdescribed by H. M. Weingartl et al, in J. Virol. Meth. 104: 203-216(2002). Weingartl described transfection of primary PAMs with a plasmidpSV3neo, carrying genes for neomycin resistance and SV40 T antigen. Thisled to the isolation of three myeloid (monocyte/macrophage) immortalizedcell lines. The outcome of this experiment however turned out to beenigmatic: no SV40 T Antigen could be detected in any of these celllines, and moreover, none of the cell lines did support PRRSVreplication. Weingartl therefore concludes that an alternativemechanism, not the presence of SV40 T Antigen, may account for theimmortalization.

After this failure, the route of using SV40 T antigen was left foralternative approaches. Yoo Jin Lee et al., found that the cell linesdeveloped by Weingartl do not express detectable levels of the 130 kDacell surface glycoprotein CD163, known to be a cellular receptor forPRRSV (J. Virol. Meth. 163: 410-415 (2010)). Thus, Yoo Jin Leeadditionally transfected one of Weingartl's PAM cell lines with theCD163 gene cloned into a retroviral vector under the control of aretroviral LTR promoter. This indeed led to the formation of animmortalized PAM cell line capable of growing PRRSV.

It was concluded in 2012 by Mingeun Sagong of the research group towhich Yoo Jin Lee belongs, that the loss of the original properties ofprimary cells such as PAM-specific markers, e.g. CD163, may be due totransformation with SV40 T antigen (J. of Virological Methods 179: 26-32(2012)). For that reason an alternative route was followed by Sagong toimmortalize PAMs. This route comprises the expression of humantelomerase reverse transcriptase (hTERT), using a retroviral vector, inPAMs to restore telomerase activity. The restoration of telomeraseactivity avoids loss of telomeric DNA and thus avoids replicativesenescence of the cells.

Mingeun Sagong concludes that their data revealed that in contrast toSV40 T antigen-transformed PAM cells, hTERT immortalization is capableof rendering the cells reliable representatives of their parental cell'sphenotype.

It follows from the above that in order to successfully transform PAMswith the aim of inducing immortalization, regardless the route followedto avoid senescence, the use of SV40 T antigen is to be avoided, and aretrovirus or at least large retroviral sequences must be used to insertthe DNA into the genome of the PAMs. A severe disadvantage of the use ofretroviruses or at least large retroviral sequences for transformingcells is that in all cases Large terminal Repeat (LTR) sequences arepresent in the DNA used for the transformation of the cells.

LTRs are retroviral elements that comprise all required signals forretroviral gene expression: enhancer, promoter, transcriptioninitiation, transcription terminator and polyadenylation signal.

These LTRs are suspected of having tumorigenic effects. This is due tothe fact that they are known to cis-activate other cellular genes andthe fact that they may recombine with other retroviral sequences in thecellular genome (Mosier, D. E., Applied Biosafety 9: 68-75 (2004)).

Nevertheless, transformation of cells with retroviral DNA comprising LTRsequences and avoiding the use of SV40 T antigen seemed to be the onlyway to obtain immortalized PAMs.

Surprisingly it was found now, that it is possible to successfullyobtain PAMs that are immortalized and still susceptible to PRRSV, andnevertheless free of retroviral Long Terminal Repeat DNA.

Such immortalized PAMs according to the invention could unexpectedly beobtained through transfection with DNA comprising SV40 T antigen, nowhowever in combination with the use of a transposon as a means to obtainstable integration in the cellular genome.

Transposons can be viewed as natural DNA transfer vehicles that, similarto integrating viruses, are capable of efficient genomic insertion.

For unknown reasons, the negative effects of SV40 T antigen with orwithout retroviral DNA as described above can unexpectedly be avoided ifcells are transformed using a DNA molecule comprising a gene encodingSV40 T antigen and transposons.

In principle, the transposons remain stably present in the cellulargenome after integration in the genome. Therefore, preferablyimmortalized PAMs according to the invention comprise transposons.

For the purpose of the present invention, an immortalized cell line is apopulation of cells (in this case PAMs) from a multicellular organismwhich would normally not proliferate indefinitely but, due to mutation,has evaded normal cellular senescence and instead can keep undergoingdivision. Such cells have escaped the normal limitation of growth foronly a finite number of division cycles.

Methods used for the preparation of an immortalised PAM according to theinvention basically comprise the following steps:

a) the step of obtaining a cell-containing bronchoalveolar lavage samplefrom a porcine subject. Such steps have been described already by i.a.Wensvoort, G. et al., in 1991 (vide supra), by Weingartl, H. M. et al.(vide supra), and by others and they are still the preferred way ofobtaining PAMs.b) the step of separating a cellular component from said sample. Thisstep is also well-known in the art, and is also described i.a. byWensvoort and by Weingartl, and it is usually done throughcentrifugation of the lung lavage material at low speed,c) the step of transfecting said cellular component with a DNA moleculecomprising transposons and comprising a gene encoding the SV40 T antigenunder the control of a suitable promoter.

Transfection can be done in many ways known in the art. Commercial kitsfor transfection are currently available through i.a. Bio-Rad (LifeScience (Research, Education, Process Separations, Food Science), LifeScience Research, 2000 Alfred Nobel Drive, Hercules, Calif. 94547, USA)and Invitrogen (Life Technology, 3175 Staley Road, Grand Island, N.Y.14072, USA). Commonly used reagent-based transfection methods comprisethe use of lipids, calcium phosphate, cationic polymers, DEAE-dextran,activated dendrimers and magnetic beads. Instrument-based methodscomprise electroporation and micro-injection.

A DNA molecule comprising transposons and comprising a gene encoding theSV40 T antigen under the control of a suitable promoter could e.g. be aplasmid comprising a gene encoding the SV40 T antigen under the controlof a suitable promoter. This plasmid may be in a circular or linear formwhen it is used for the transfection step.

The use of transposons as such is well-known in the art. A paper byIvics, Z. and Izsvak Z. extensively reviews transposons and their use,and provides insight in the mechanisms of action of transposons (MobileDNA 1: 25-39 (2010)).

A review paper by Deepika Ahuja et al., about SV40 T antigen providesinsight in the mechanisms of action of this protein (Oncogene 24:7729-7745 (2005)). Basically, SV40 T antigen inhibits the p53 andRb-family of tumor suppressors. It is this activity of the T antigenthat is thought to cause transformation of the cells towards theirimmortalized character.

A large number of suitable promoters for the expression of the SV40 Tantigen are known in the art, which are recognized for their efficientlevel of expression. They include classic promoters such as the (human)cytomegalovirus immediate early promoter (Seed, B. et al., Nature 329,840-842, 1987; Fynan, E. F. et al., PNAS 90, 11478-11482, 1993; Ulmer,J. B. et al., Science 259, 1745-1748, 1993), the Human Cytomegalovirusenhancer-promoter for the expression of gD of BoHV-1. (Donofrio G., etal., Clinical and Vaccine Immunology 13: 1246-1254, (2006)), the MouseCytomegalovirus immediate early (MCMViel) promoter, the MouseCytomegalovirus early (MCMVel) promoter, SV40 immediate early promoter(Sprague J. et al., J. Virology 45, 773, 1983), the SV-40 promoter(Berman, P. W. et al., Science, 222, 524-527, 1983), the metallothioneinpromoter (Brinster, R. L. et al., Nature 296, 39-42, 1982), the heatshock promoter (Voellmy et al., Proc. Natl. Acad. Sci. USA, 82, 4949-53,1985), the major late promoter of Ad2 and the β-actin promoter (Tang etal., Nature 356, 152-154, 1992).

A preferred promoter is the CAG promoter. (Miyazaki, J; Takaki, S;Araki, K; Tashiro, F; Tominaga, A; Takatsu, K; Yamamura, K., Gene 79(2): 269-77 (1989), and Niwa, H; Yamamura, K; Miyazaki, J. Gene 108 (2):193-9 (1991).)

d) the step of selecting cells that are capable of sustainedproliferation.

PAM cells that are capable of sustained proliferation are cells thathave been cultured for at least 5 cell cycles. The cell cycle, orcell-division cycle, is the series of events that take place in a cellleading to its division and duplication (the cell replication). Theselection of cells that are capable of sustained proliferation is a verysimple process for the following reason: primary PAMs are hardly or notcapable of dividing outside their natural environment; the porcine lung.As can be seen from FIG. 2, first 2 bars (no M-CSF added) the number oflive primary PAM cells after lung lavage and isolation decreases overtime. In a culture starting with 200000 PAM cells, only about half ofthe cells are still viable after 3 days. This amount further decreasessteadily over time.

This means that if there is an increase in the number of cells, thismust be due to the fact that one or more cells have successfully beentransfected with the DNA molecule comprising the transposon and the geneencoding the SV40 T antigen is inserted in the cellular genome. Sobasically the process is self-selecting: maintenance of PAMs that weresuccessfully transformed in a suitable cell growth medium willautomatically lead to replication of successfully transformed cells,whereas non-immortalised cells will stop dividing and die off. Suitablecell growth media are known in the art and are described i.a. in theExamples section. They are also described i.a. by Wensvoort, G. et al.,in 1991 (vide supra), by Weingartl, H. M. et al. (vide supra). Furtherguidance about cell culture conditions can be found in the Examples.

Thus, one embodiment of the present invention relates to a method forthe preparation of an immortalised PAM, wherein that said methodcomprises the steps of

-   -   a) obtaining a cell-containing bronchoalveolar lavage sample        from a porcine subject,    -   b) separating a cellular component from said sample,    -   c) transfecting said cellular component with a DNA molecule        comprising transposons and comprising a gene encoding the SV40 T        antigen under the control of a promoter, and    -   d) selecting cells that can be cultured for at least 5 cell        cycles

Usually, cells are selected that have been cultured for at least 5 cellcycles. For such cells it can reasonably be assumed that they aresuccessfully immortalized PAMs, since primary PAMs will usually notreplicate more than one or two times, exceptionally up to 5 times, invitro after isolation from the lungs.

In exceptional cases, early cell cycles may show instable behavior, e.g.due to the fact that the transposon has integrated in the cellulargenome at a very critical site, or due to instable integration of thegene encoding the SV40 T antigen. Therefore, in practice cells areselected that have been cultured for at least 10, 15, 20, 25, 30, 40, 50or even 60 cell cycles in that order of preference.

The chances of any instability becoming manifest do decrease with theamount of cell cycles of the selected immortalised PAM.

Thus, preferably, cells are selected that have been cultured for atleast 10, 15, 20, 25, 30, 40, 50 or even 60 cell cycles in that order ofpreference.

The presence of Macrophage Colony Stimulating Factor (M-CSF) does notsignificantly stimulate replication of primary PAMs for more than a fewcell divisions.

The presence of granulocyte-M-CSF (gM-CSF) may improve the condition ofthe primary PAMs, even to the extent that there is some replication fora very short period of time. It was however shown by the inventors thatthe use of gM-CSF leads to a decrease in CD163-expression. And sinceCD163 is involved in the replication of PRRSV to PAMs, the use of gM-CSFmay in this respect not have a nett beneficial effect.

M-CSF appears to improve the condition of the primary PAMs to a lesserextent than gM-SCF, but it does not interfere with CD163-production.

It was however surprisingly found that the presence of M-CSF in thegrowth medium of PAMs obtained through lung lavage, before they aresubjected to transfection, makes the cells somewhat better resistant tothe stressful process of transfection. Thus, the efficiency oftransfection is significantly increased in the presence of M-CSF.

Suitable amounts of M-CSF are e.g. 5, 10, 25, 50, 100 or 200 ng/ml inthat order of increasing order of preference.

(In FIG. 2 it can be seen that indeed in the presence of M-CSF, thenumber of live primary PAMs cells after lung lavage and isolationdecreases (or at best stays stable for 6 days). The decrease is lessdramatic over time when compared to the decrease in the absence ofM-CSF, but in any case there is within the statistical probability noincrease in the number of cells).

Therefore, another preferred form of this embodiment relates to methodsaccording to the invention, wherein the method comprises the step ofadding an amount of at least 5 ng/ml of M-CSF to the cell-containingbronchoalveolar lavage sample and/or the cellular component before thetransfection step. PCT Patent Application WO2008/089094 discloses theuse of M-CSF as a mandatory growth medium component in order to keepimmortalized but non-transformed fetal PAMs alive. Contrary to this, inthe present invention M-CSF or gM-CSF is used before the PAMs areimmortalized.

It was shown by the inventors that unexpectedly the presence of M-CSFduring step d) and/or while culturing immortalised PAMs according to theinvention, i.e. transformed and non-fetal PAMs, also improves theviability of these transformed and non-fetal cells according to theinvention. It can be seen in FIG. 4, that both the viability andreplication rate of immortalized PAMs according to the inventionimproves significantly in the presence of M-CSF. Small amounts of M-CSFof a magnitude of 1, 2, 3, 4 or 5 ng/ml already suffice to improve bothviability and replication rate. Preferred concentrations of M-CSF are 6,12, 25, 50, 100, 200 or even 400 ng/ml, in increasing order ofpreference.

Thus, again another preferred form of this embodiment relates to amethod according to the invention wherein the method additionallycomprises the step of adding an amount of at least 1 ng/ml of M-CSFduring step d) and/or while culturing the immortalised PAM according tothe invention.

FIG. 5 shows that antibodies against CD163 and P210, two receptors thatwere demonstrated to be essential for entry and replication of PRRSvirus in PAM cells, are indeed reactive with immortalized PAMs accordingto the invention. This means that CD163 and P210 are indeed present onimmortalized PAMs according to the invention.

FIG. 6 shows that immortalized PAMs according to the invention indeedsupport PRRSV replication. It can be seen, that a PRRSV field isolatereplicates even faster and to a higher titer in the first 2-3 days afterinfection, when compared with primary PAMs.

FIG. 7 shows, that indeed a PRRSV field isolate replicates faster and toa higher titer on immortalized PAMs according to the invention, whencompared to replication on MARC-145 cells. It can also be seen that afield isolate indeed replicates better on PAMs in general, regardless ifthey are immortalized or not, when compared to replication on MARC-145cells.

FIG. 7 also shows that vice versa PRRSV Type I and Type II strains thatare adapted to replication on MARC-145 cells replicate to a higher titeron MARC-145 cells when compared to replication on PAMs.

This shows that indeed immortalized PAMs according to the invention areas suitable as primary PAMs to replicate PRRSV field isolates, and arethus more suitable to this end than non-PAM cells such as MA104,MARC-145 or CL2621 cells.

A second embodiment of the present invention relates to an immortalisedporcine alveolar macrophage (PAM), characterized in that the PAM issusceptible to Porcine Respiratory and Reproductive Virus (PRRSV), thePAM expresses an SV40 T antigen and the PAM does not comprise retroviralLong Terminal Repeat DNA.

Immortalised PAMs according to the invention can in principle further beprovided with a functional gene encoding human telomerase reversetranscriptase (hTERT). This is however by no means necessary, since theSV40 T antigen is capable of maintaining the immortalized status of thePAMs according to the invention. In fact, if only for technicalsimplicity of producing immortalised PAMs, it is preferred that onlySV40 T antigen is used for the immortalization of the cells.

Thus, a preferred form of this embodiment relates to immortalised PAMsaccording to the invention that are characterized in that the PAMs donot comprise hTERT.

A third embodiment of the present invention relates to methods ofreplicating PRRS virus, characterized in that such methods comprise thesteps of

a) culturing an immortalised PAM according to the invention,b) contacting the immortalised PAM with the PRRSc) allowing the PRRSV to replicate andd) isolating the progeny virus.

A fourth embodiment of the present invention relates to a cell culturecomprising an immortalised PAM according to the invention.

In a preferred form of this embodiment, the cell culture comprising theimmortalised PAM is infected with PRRSV.

In another preferred form of this embodiment, the cell culturecomprising the immortalised PAM comprises M-CSF.

A fifth embodiment of the present invention relates to methods for thepreparation of a vaccine comprising PRRSV, characterized in that themethods comprise the method of replicating PRRSV according to theinvention followed by the step of mixing the virus with apharmaceutically acceptable carrier.

In a preferred form of this embodiment, the PRRSV is in a liveattenuated or an inactivated form.

EXAMPLES Example 1 Materials and Methods Plasmids.

To construct pPB-CAG-SV40 T Ag, XhoI and BIII sites were added to SV40 TAg by PCR using primers SV40 Tag 5′-BII(5′-GGCGAGATCTACCATGGATAAAGTTTTAAACAG-3′) and SV40 Tag 3′-XI(5′-GGCGCTCGAGTTATGTTTCAGGTTCAGGGG-3′). Phusion DNA polymerase was usedfor PCR according to the manufacturer's protocol (New England Biolabs).The fragment was cloned into pCR-Blunt (Life Technologies) and verifiedby sequencing. Next, SV40 T Ag was excised from pCR-Blunt and clonedinto pPB-CAG-EBNXN (Yusa et al., 2009) using the BIII-XhoI sites tocreate pPB-CAG-SV40 T Ag (FIG. 1). The final construct was verified bysequencing. Plasmid DNA for transfection into primary PAM cells wasisolated with the Qiagen EndoFree plasmid maxi kit (Qiagen).

Isolation and Growth of Primary Cells and PAM SVh Cells.

Porcine alveolar macrophages were harvested from the lungs of 1-2 weekold PRRSV-negative, SPF piglets. The lungs were washed three to fivetimes with sterile phosphate-buffered saline (PBS) solution. The washingfluid was centrifuged 10′ at 1000×g at 4° C. to pellet cells. Cells wereresuspended and stored in liquid nitrogen in RPMI 1640+HEPES+GlutaMax(Life Technologies) containing 50% FCS (Hyclone, Thermo Scientific), lxnon-essential amino acids (Life Technologies), 2 mM glutamine,antibiotics and 10% DMSO. Upon thawing, PAM cells were taken intoculture and grown in RPMI 1640+HEPES+GlutaMax (Life Technologies)containing 20% FCS (Hyclone, Thermo Scientific), 1× non-essential aminoacids (Life Technologies), 2 mM glutamine, antibiotics at 37° C. and 5%CO₂. Recombinant human M-CSF (M-CSF) was purchased from R&D Systems. PAMSVh cells were grown in medium+100 ng/ml M-CSF (R&D Systems).

Viability Assays.

The effect of M-CSF on in vitro survival of primary PAM cells wasexamined by seeding 200.000 cells per 24-well in medium containingdifferent concentrations of M-CSF. Each condition was tested in duplo.Cell samples were taken from the wells 3 and 6 days after seeding andthe number of viable cells was determined with the GUAVA Easycyte plus(Guava Millipore) using Viacount dye (Guava Millipore) according to themanufacturer's protocol. Each sample was counted twice.

The effect of M-CSF concentration on PAM SVh proliferation was examinedin similar fashion with minor adjustments. Here, 25000 cells were seededin ultra-low attachment 96-well plates and cells were harvested forcounting 3, 4, 5 and 6 days after seeding. Each sample was countedtwice.

Transfection.

After 6 days in culture, primary PAM cells were harvested and viablecells were counted. In this experiment M-CSF (100 ng/ml) was added tothe medium to promote in vitro survival of primary PAMs. Pertransfection, 1.10E6 viable cells were transfected in 100 μl Primarycell buffer P3+ supplement (Lonza Cologne AG) using program DN-100 ofthe Nucleofector 4D (Lonza Cologne AG). Cells were either transfectedwith 1.6 μg pPB-CAG-SV40 T Ag and 0.4 μg pPB-CMV-hyPBase (Yusa et al.,2011) or, as a control, with 1.6 μg pPB-CAG-EBNXN and 0.4 μgpPB-CMV-hyPBase. After administration of the Nucleofection pulse, cellswere left at RT for 10 min. Next, 400 μl RPMI 1640 (37° C.) was slowlyadded to the cells and cells were incubated at 37° C. for 5 minutes.Then, cells were carefully resuspended, seeded in RPMI1640+HEPES+GlutaMax (Life Technologies) containing 20% FCS (Hyclone,Thermo Scientific), 1× non-essential amino acids (Life Technologies), 2mM glutamine, antibiotics and 100 ng/ml M-CSF (R&D Systems) andincubated at 37° C. and 5% CO2.

Antibodies and Flow Cytometry.

Cells were labeled with mouse monoclonal antibodies raised againstporcine CD163 (clone 2A10/11, AbD Serotec), mouse monoclonal antibodiesraised against porcine sialoadhesin/p210 (Duan et al., 1998) orFITC-labeled mouse IgG1 isotype control antibodies (AbD Serotec). Afterwashing, cells labeled with anti-CD163 or anti-sialoadhesin/p210antibodies were labeled with FITC-labeled goat-anti-mouse antibodies(Lifespan Biosciences). Cells were analyzed using a Becton DickinsonFACS Calibur cytometer and CellQuest Pro software.

PRRSV Replication and Titration.

To compare primary PAMs, PAM SVh or MARC-145 cells as substrates forPRRSV replication, equal amounts of cells were seeded in 12-wells. Cellswere infected at t=0 with either a pathogenic field isolate, a Type Ivaccine strain or a Type II vaccine strain with MOI 0,001 or MOI 0,0001.Supernatants were harvested several days after infection and stored at−20° C. Virus titers were determined by titrating primary PAM and PAMSVh supernatants on primary PAMs and MARC-145 supernatants on MARC-145cells. All titrations were performed in duplo. Titers were calculatedusing the method of Spearman-Kärber and expressed as log₁₀TCID₅₀/ml.

Results: M-CSF Promotes In Vitro Viability of Primary PAMs.

Primary PAMs have a low in vitro survival rate in standard RPMI 1640medium containing 20% FCS (FIG. 2). The number of viable cells declinesin time and only about 50% of cells is still viable after three days.Addition of macrophage-colony stimulating factor (M-CSF) to the culturemedium has a positive effect on survival and clearly increases thenumber of viable cells after three or six days compared to cells grownin absence of M-CSF.

Establishment of an SV40-Immortalized PAM Cell Line.

Primary cells were grown for 6 days in medium with M-CSF andsubsequently transfected with pPB-CAG-SV40 T Ag or pPB-CAG-EBNXN incombination with the pPB-CMV-hyPBase vector encoding the piggyBactransposase. After transfection, cells were carefully monitored each dayfor proliferation and the medium was replenished regularly with freshmedium+100 ng/ml M-CSF. No cell proliferation was visible in thepPB-CAG-EBNXN transfected control cells and 4-5 weeks after transfectionall cells were dead. In contrast, small colonies grew out in thecultures of pPB-CAG-SV40 T Ag transfected cells 3-4 weeks aftertransfection (FIG. 3). These colonies continued to proliferate and werepassaged to increase cell number. All cells were SV40 T Ag positive asdemonstrated by immunofluorescence (data not shown).

These cells continue to proliferate, can be passaged twice a week andhave currently been kept in culture for more than 8 months (50-60passages). This cell line can easily be regrown in culture after liquidnitrogen storage. The thus established cell line was named PAM SVh.

Proliferation of PAM SVh Cell Line Depends on M-CSF-Concentration.

To determine whether the PAM SVh cell line requires M-CSF forproliferation, PAM SVh cells were grown without or in the presence ofdifferent concentrations of M-CSF. The number of viable cells wasdetermined 3 and 6 days after seeding. Proliferation of PAM SVh isM-CSF-dependent in a concentration-dependent manner (FIG. 4). Thelargest increase in cell number is seen in the presence of highconcentrations (400-100 ng/ml) of M-CSF. Lower concentrations of M-CSFresult in reduced proliferation of cells and little or no increase incell number was detected in the absence of M-CSF, indicating thatproliferation of PAM SVh cells depends on M-CSF concentration in themedium.

PAM SVh Cells Express Sialoadhesin/p210 and CD163 Markers.

Two receptors have been demonstrated to be essential for entry andreplication of PRRS virus in PAM cells, sialoadhesin/P210 and CD163.Whereas expression of sialoadhesin/p210 was found to be essential forbinding and entry of PRRSV (in)to PAM cells, CD163 was shown to berequired for PRRSV replication in cells (Delputte et al., 2005; Van Gorpet al., 2008; Calvert et al., 2007). We examined whether PAM SVh cellsexpressed sialoadhesin/P210 and CD163 by labeling cells with specificantibodies raised against these receptors and analyzing them by flowcytometry. More than 80% of PAM SVh cells were found to be CD163+ andmore than 70% sialoadhesin/P210+(FIG. 5), suggesting that these cellsmight be suitable for infection with and replication of PRRSV.

PAM SVh Cells are Suitable Substrates for PRRSV Replication.

We tested whether PAM SVh cells are a substrate for PRRSV replication byinfecting them with a pathogenic field isolate. Supernatants wereharvested at different days after infection and titrated to determinevirus titers. For comparison, we also infected primary PAM cells in thesame experiment. PAM SVh cells were infected by the PRRSV field isolateand clearly produce PRRSV virus (FIG. 6). Compared to primary PAMs,virus titers produced by PAM SVh cells were higher at day 1 and day 2after infection, comparable at day 3 and day 4 and lower at day 5.

MARC-145 cells are commonly used as substrate for production of PRRSVvaccine strain viruses. We compared primary PAMs, PAM SVh cells andMARC-145 cells as substrates for replication of different PRRSV strains.We infected equal numbers of primary PAMs, PAM SVh and MARC-145 cellswith either a pathogenic field isolate, a PRRSV Type I vaccine strain ora PRRSV Type II vaccine strain. Supernatants were harvested at differentdays after infection and supernatants were titrated to determine virustiters. Again, PAM SVh cells produce comparable or higher titers of thePRRSV field isolate than primary PAMs (FIG. 7A). PAM SVh also producehigher titers of the PRRSV field isolate than MARC-145 cells. When wecompared virus titers of the PRRSV Type I and II strains produced on thedifferent substrates, we found that both strains replicated best onMARC-145 cells, the substrate which is normally used for production ofthese attenuated viruses (FIGS. 7B and C). PAM SVh cells, however,produced higher titers than primary PAMs for both vaccine strains at alltime-points, again demonstrating that PAM SVh cells are a bettersubstrate for PRRSV replication than primary PAMs.

LEGEND TO THE FIGURES

FIG. 1: Vector map pPB-CAG-SV40 T Ag

FIG. 2: M-CSF increases in vitro survival of primary PAMs. 200000 cellsprimary PAM cells were seeded in duplo at t=0 (day 0) in mediumcontaining no or different concentrations of M-CSF. The number of viablecells was determined at 3 and 6 days after seeding. Cell numbers weredetermined in duplo per well. Data depicted are mean+SEM of fourindependent measurements.

FIG. 3: Colony formation in pPB-CAG-SV40 T Ag transfected cells Coloniesare indicated by black arrows.

FIG. 4: M-CSF stimulates proliferation of PAM SVh cells. 25000 cells PAMSVh cells were seeded at t=0 (day 0) in medium containing no ordifferent concentrations of M-CSF. The number of viable cells wasdetermined 3, 4, 5 and 6 days after seeding. Data depicted are mean oftwo cell counts per well.

FIG. 5: PAM SVh cells express CD163 and sialoadhesin. PAMSVh cells werelabeled with antibodies raised against CD163 or p210 or isotype controlantibodies. Cells were labeled with FITC-labeled secondary antibodiesand analyzed by flow cytometry. The percentage of FITC-positive cellsper antibody is depicted.

FIG. 6: PRRSV replication on PAM SVh cells. Cells were infected at t=0with a pathogenic PRRSV field isolate (MOI 0,001). Supernatants wereharvested at different days after infection and titrated to determinevirus titers. The 10 log values of the TCID50/ml are depicted forprimary PAMs (open bars) and PAM SVh (solid bars). Data are mean of twoindependent titrations.

FIG. 7: Replication of different PRRSV strains on PAM SVh cell line.Cells were infected at t=0 with either (A) a pathogenic PRRSV fieldisolate (MOI 0,0001), (B) PRRSV Type I vaccine strain (MOI 0,001) or (C)PRRSV Type II vaccine strain (MOI 0,001). Supernatants were harvested atdifferent days after infection and titrated to determine virus titers.The 10 log values of the TCID50/ml are depicted for MARC-145 (openbars), primary PAMs (shaded bars) and PAM SVh (solid bars). Data aremean of two independent titrations

REFERENCE LIST

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1. An immortalized porcine alveolar macrophage (PAM), wherein the PAM issusceptible to Porcine Respiratory and Reproductive Virus (PRRSV),expresses an SV40 T antigen and does not comprise retroviral LongTerminal Repeat DNA capable of retroviral gene expression.
 2. Theimmortalized PAM according to of claim 1, wherein the PAM does notcomprise retroviral Long Terminal Repeat DNA in use for the expressionof the SV 40 T antigen.
 3. The immortalized PAM of claim 1, wherein thePAM comprises transposons.
 4. The immortalized PAM according to any ofclaim 1, wherein the PAM does not comprise human telomerase reversetranscriptase (hTERT).
 5. A cell culture comprising the immortalized PAMaccording of claim
 1. 6. A cell culture comprising the immortalized PAMof claim 1, wherein the cell culture is infected with PRRSV.
 7. A cellculture comprising the immortalized PAM of claim 1, wherein the cellculture comprises M-CSF.
 8. A method for preparing an immortalizedporcine alveolar macrophage (PAM) that is susceptible to PorcineRespiratory and Reproductive Virus (PRRSV), expresses an SV40 T antigen,and does not comprise retroviral Long Terminal Repeat DNA capable ofretroviral gene expression; wherein said method comprises the steps of:a) obtaining a cell-containing bronchoalveolar lavage sample from aporcine subject, b) separating a cellular component from said sample, c)transfecting said cellular component with a DNA molecule comprisingtransposons and comprising a gene encoding the SV40 T antigen under thecontrol of a suitable promoter, and d) selecting PAM cells that havebeen cultured for at least 5 cell cycles.
 9. The method of claim 8,wherein PAM cells are selected that have been cultured for at least 10cell cycles.
 10. The method of claim 8, wherein said method comprisesthe step of adding an amount of at least 5 ng/ml of M-CSF to thecell-containing bronchoalveolar lavage sample, the cellular component,or both the cell-containing bronchoalveolar lavage sample and thecellular component before the transfection step.
 11. The method of claim8, wherein said method additionally comprises the step of adding anamount of at least 1 ng/ml of macrophage colony stimulating factor(M-CSF) during step d) or while culturing the immortalized PAM.
 12. Amethod of replicating PRRSV, said method comprising the steps of: a)culturing an immortalized PAM of claim 1, b) contacting the immortalizedPAM with the PRRS virus, c) allowing the PRRSV to replicate, and d)isolating the progeny virus.
 13. A method for the preparation of avaccine comprising PRRSV, wherein the method comprises following themethod of replicating PRRSV of claim 12 followed by the step of mixingthe replicated PRRS virus with a pharmaceutically acceptable carrier.14. The method of claim 13, wherein the PRRSV is in a live attenuatedform.
 15. The method of claim 13, wherein the PRRSV is in an inactivatedform.
 16. The method of claim 8, wherein said method additionallycomprises the step of adding an amount of at least 1 ng/ml of macrophagecolony stimulating factor (M-CSF) during step d) and while culturing theimmortalized PAM.
 17. The immortalized PAM of claim 2, wherein the PAMcomprises transposons.
 18. The immortalized PAM of claim 17, wherein thePAM does not comprise human telomerase reverse transcriptase (hTERT).19. A cell culture comprising the immortalized PAM of claim
 18. 20. Acell culture comprising the immortalized PAM of claim 17.